Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, amplification and sequencing require that nucleic acids used in the techniques be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. substances that block or inhibit nucleic acid hybridizations, enzymatically catalyzed reactions and other types of reactions used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances which block or mask detection of the nucleic acid of interest. Substances of this last type can block or mask by providing a "background" indicative of the presence in a sample of a quantity of a nucleic acid of interest, (also referred to herein as a "target nucleic acid") when the nucleic acid of interest is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a target nucleic acid is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors, oligonucleotides, or non-target nucleic acids. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining target nucleic acid sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which the target nucleic acid is typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the target nucleic acid of interest must be isolated before being used in a molecular biological procedure.
The earliest techniques developed for use in isolating target nucleic acids from such complex systems typically involve multiple organic extraction and precipitation steps. Hazardous chemicals, such as chloroform and phenol or mixtures thereof, were used in most such procedures. Closed circular nucleic acid molecules, such as plasmid DNA, was typically isolated further by ultra-centrifugation of plasmid DNA in the presence of cesium chloride and ethidium bromide. See, e.g., Molecular Cloning, ed. by Sambrook et al. (1989), pp. 1.42-1.50. Ethidium bromide is a neurotoxin. Removal of both ethidium bromide and cesium chloride from the resulting band of plasmid DNA obtained by ultracentrifugation was required before the DNA could be used in downstream processing techniques, such as sequencing, transfection, restriction analysis, or the polymerase chain reaction.
In recent years, many different matrices have been developed for use in the isolation of nucleic acids from complex biological materials. For example, matrices have been developed for the isolation of nucleic acids by ion-exchange chromatography (e.g., J. of Chromatog. 508:61-73 (1990); Nucl. Acids Research 21(12):2913-2915 (1993); U.S. Pat. No.'s 5,856,192; 5,82,988; 5,660,984; and 4,699,717), by reversed phase (e.g. Hirbayashi et al., J of Chromatog. 722:135-142 (1996); U.S. Pat. No. 5,057,426, by affinity chromatography (e.g., U.S. Pat. No. 5,712,383; and PolyATract.RTM. mRNA Purification System (Promega Corp., Madison, Wis.; see Promega's Technical Manual No. TM031), and by matricies which employ a combination of the above isolation modes (see, e.g. U.S. Pat. No. 5,652,348; J. Chromatography 270:117-126(1983))
One of the first solid phases developed for use in isolating nucleic acids was a specialized resin of porous silica gel particles designed for use in high performance liquid chromatography (HPLC). The surface of porous silica gel particles was functionalized with anion-exchangers which could exchange with plasmid DNA under certain salt and pH conditions. See, e.g. U.S. Pat. Nos.: 4,699,717, and 5,057,426. Machrey-Nagel Co. (Duren, Germany) was one of the first companies to provide HPLC columns packed with such anion-exchange silica gel particles, and it continues to sell such columns today. See, e.g. Information about NUCLEOGEN.RTM. 4000-7DEAE in product information downloaded from the Machrey-Nagel homepage on the Internet on Jun. 12, 1998, at http://www.machrey-nagel.com. Each such column was designed so that plasmid DNA bound thereto is eluted in an aqueous solution containing a high concentration of a highly corrosive salt (e.g. plasmid DNA is eluted from the NUCLEOGEN.RTM. 4000-7DEAE column in 6 M urea). Each such column had to be washed thoroughly between each isolation procedure to remove the corrosive salt and contaminants bound to the column with the DNA from the system. The nucleic acid solution eluted therefrom also had to be processed further to remove the corrosive salt therefrom before it could be used in standard molecular biology techniques, such as cloning, transformation, digestion with restrictive enzymes, or amplification.
Various silica-based solid phase separation systems have been developed since the early HPLC systems described above. (See, e.g. the silica gel and glass mixture for isolating nucleic acids according to U.S. Pat. No. 5,658,548, and the porous support with silane bonded phase used to isolate oligonucleotides according to U.S. Pat. No. 4,767,670.) Modern silica-based systems utilize controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, glass fibers or mixtures of the above. Each modern silica-based solid phase separation system is configured to reversibly bind nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. Such solid phases are designed to remain bound to the nucleic acid material while the solid phase is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the solid phase by exposing the solid phase to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems. See, e.g. Wizard.RTM. DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.); or the QiaPrep.RTM. DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.)
Magnetically responsive particles, formerly used to isolate and purify polypeptide molecules such as proteins or antibodies, have also been developed for use as solid phases in isolating nucleic acids. Several different types of magnetically responsive particles designed for isolation of such materials are described in the literature, and many of those types of particles are available from commercial sources. Such particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to reversibly bind nucleic acid materials through an intermediary. For an example of particles of the first type, see silica based porous particles designed to reversibly bind directly to DNA, such as MagneSil.TM. particles from Promega, or BioMag.RTM. magnetic particles from PerSeptive Biosystems. For examples of particles and systems of the second type designed to reversibly bind one particular type of nucleic acid (mRNA), see the PolyATract.RTM. Series 9600.TM. mRNA Isolation System from Promega Corporation (Madison, Wis., U.S.A.); or the ProActive.RTM. line of streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Ind., U.S.A.). Both of these latter two systems employ magnetically responsive particles with avidin subunits covalently attached thereto, and streptavidin with an oligo dT moiety covalently attached thereto. The streptavidin-oligo dT molecules act as intermediaries, hybridizing to the poly A tail of mRNA molecules when placed into contact therewith, then binding to the particles through a releasable streptavidin-avidin bond.
The indirect binding magnetic separation systems for nucleic acid isolation or separation all require at least three components, i.e. magnetic particles, an intermediary, and a medium containing the nucleic acid material of interest. The intermediary/nucleic acid hybridization reaction and intermediary/particle binding reaction often require different solution and/or temperature reaction conditions from one another. Each additional component or solution used in the nucleic acid isolation procedure adds to the risk of contamination of the isolated end product by nucleases, metals, and other deleterious substances.
Various types of magnetically responsive silica based particles have been developed for use as solid phases in direct or indirect nucleic acid binding isolation methods. One such particle type is a magnetically responsive glass bead, preferably of a controlled pore size. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tends to bind very tightly to glass, however, so that it can be difficult to remove once bound thereto. Therefore, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica.
Another type of magnetically responsive particle designed for use as a solid phase in direct binding and isolation of nucleic acids, particularly DNA, is a particle comprised of agarose embedded with smaller ferromagnetic particles and coated with glass. See, e.g. U.S. Pat. No. 5,395,498. A third type of magnetically responsive particle designed for direct binding and isolation of nucleic acids is produced by incorporating magnetic materials into the matrix of polymeric silicon dioxide compounds. See, e.g. German Patent No. DE 43 07 262 A1. The latter two types of magnetic particles, the agarose particle and the polymeric silicon dioxide matrix, tend to leach iron into a medium under the conditions required to bind nucleic acid materials directly to each such magnetic particle. It is also difficult to produce such particles with a sufficiently uniform and concentrated magnetic capacity to ensure rapid and efficient isolation of nucleic acid materials bound thereto.
Silica-based solid phase nucleic acid isolation systems, whether magnetic or non-magnetic based or configured for direct or indirect binding, are quick and easy to use and do not require the use of corrosive or hazardous chemicals. However, such are ineffective at isolating nucleic acids from contaminants, such as endotoxins, which tend to bind to and elute from such solid supports under the same conditions as nucleic acids. See, e.g. Cotten, Matt et al. Gene Therapy (1994) 1:239-246.
Some nucleic isolation systems have been developed in which a nucleic acid solution containing proteins is pre-treated with proteases to digest at least some of the proteins contained therein prior to isolation of the nucleic acid using a silica-based solid support of the type described above. See, e.g. QiaAmp.TM. Blood Kit provided by QIAGEN Inc. (Santa Clarita, Calif.), which utilizes protease; and Wizard.RTM. Plus SV Minipreps DNA Purification System provided by Promega Corp. (Madison, Wis.), which utilizes an alkaline protease. However, such pre-treatment systems require the introduction of one contaminant into a mixture to digest another contaminant. Carry-overproteases can limit the utility of nucleic acids isolated using such modified silica-based systems at least as much as nucleic acid samples contaminated with the proteins the proteases are introduced to digest. Specifically, given the proper solution conditions, proteases in a nucleic acid solution will digest any proteins introduced into the solution, including enzymes introduced therein to modify, cut, or transcribe the nucleic acid contained therein for downstream processing or analysis. Protease addition, incubation and removal steps also drive up the cost of nucleic acid isolation, costing time and money compared to isolation systems with no such additional steps.
In all the solid phase systems described above, each solid phase used therein has a substantially uniform surface composition designed to bind to a nucleic acid of interest, in the form of a silica or silica gel surface, or in the form of a silica gel or polymer surface modified with chemical groups exhibiting anion exchanger activities. Bimodal and multimodal systems have also been developed, such as systems: (1) in which multiple columns each of which contains a solid phase modified with a different chemical group from the other columns in the system (e.g., Wheatley J. B., J. Chromatogr. (1992) 603: 273); (2) in which a single column is used with a single solid phase with at least two different chemical groups (e.g., Patent '680; Little, E. L. et al., Anal. Chem. (1991) 63: 33); or (3) in which two different solid phases are employed in the same column, wherein the two solid phases are separated from one another within the column by solid porous dividers (e.g., U.S. Pat. No. 5,660,984). Each of the chemical groups on the surface of the solid supports in the single column or multicolumn multimodal systems is configured to bind to different materials in whatever substrate is introduced into the system. Only a few such bimodal or multimodal column chromatography systems have been developed specifically for nucleic acid isolation (see, e.g. U.S. Pat. No. 5,316,680). Surface group combinations used in such solid phase systems include reverse phase, ion exchange, size exclusion, normal phase, hydrophobic interaction, hydrophilic interaction, and affinity chromatography. Such systems are designed such that only one of the surface groups binds a target species, such as a nucleic acid, while the other surface group(s) bind to and remove one or more non-target species in a mixture.
Bimodal and multimodal systems are far from simple, efficient alternatives to conventional organic or resin methods of nucleic acid isolation described above. Multi-column systems are inherently complex to run, as each column requires a unique set of mobile phase conditions to bind and/or release the desired target or non-target species bound to the stationary solid phase of the system. Non-target species tend to block adjacent functional groups configured to bind to the target species, thus adversely affecting overall yield. Also, all the bimodal or multimodal systems are only designed to separate a target species from other species for which functional groups have affinity.
At least one mixed mode ion exchange solid phase system has been developed for use in isolating certain types of target compounds, such as proteins or peptides, from an aqueous solution. See U.S. Pat. No. 5,652,348 (hereinafter, "Burton et al. '348") at col. 4, lines 21 to 25. The mixed mode ion exchange system of Burton et al. '348 comprises a solid support matrix with ionizable ligands covalently attached to the sold support matrix. The ionizable ligand is capable of exchanging with and adsorbing the target compound at a first pH and of releasing or desorbing the target compound at a second pH. The ionizable functionality is "either further electrostatically charged or charged at a different polarity at the second pH". (Burton et al. '348, claim 1, col. 25, lines 46-50). The examples of mixed mode ion exchange solid phase systems provided in the Burton et al. '348 patent contain only a single ionizable functionality, an amine residue capable of acting as an anion exchange group at the first pH. The concentration of ionizable ligands present on the solid support matricies disclosed in Burton et al. '348 is sufficiently high to "permit target protein binding at both high and low ionic strength". The only ligand density specifically disclosed and claimed as sufficiently high for the mixed mode ion exchange solid phase of Burton et al. '348 to bind to target proteins at high and low ionic strength is a ligand density which is "greater than the smaller of at least about 1 mmol/gram dryweight of resin or at least about 150 .mu.mol/ml of resin" (col 13, lines 22-23; and claim 1). The mixed mode ion exchange system of Burton et al. '348, is specifically designed for use in the isolation of proteins and peptides, not nucleic acids or oligonucleotides.
Materials and methods are needed which can quickly, safely, and efficiently isolate target nucleic acids which are sufficiently free of contaminants to be used in molecular biology procedures. The present invention addresses the need for materials and methods which provide a rapid and efficient means for isolating target nucleic acids from any mixture of target nucleic acids and contaminants, including lysates of gram-negative bacteria, thereby providing purified nucleic acids which can be used in a variety of biological applications, including transfection of cultured cells and in vivo administration of nucleic acids to organisms.